Team:Paris/6 August 2009

protocol n°2 (05/08/09) but using PBS

->nearly the same as MgSO4

protocol n°2 (05/08/09)but 1ml of cells and 1µl of dye, one with MgSO4 and one with PBS

->not enough dye

• [Ladder 1kb|A1|nothing|A2|nothing|A3]using 1% agarose during 30min
  • A1 = Tg3p = 274bp (utiliser le ladder 100bp)
  • A2 = TE3 = 1036bp
  • A3 = ClyA (with RBS and poly G linker = 987bp

• A1 and A2 didn't work we don't see it on the gel because it's not in E.Coli.
• A3 didn't migrate very well
  • [Ladder 1kb|nothing|A5|nothing|A5|nothing] using 1% agarose during 30min
  • A5 = TolRII = 279bp

• Purification of A3, A4 directly in micro-column, A5, A6 using gel then micro-column
• Digestion with XbaI and PSTI

• New Miniprep for P1: pSB2K3, P13: pSB1A3, P14: BBa_K136050 (RBS tetR)
• Result: Just good for trash. Make new ON culture.

• P1: pSB2K3 by XP
• P2: BBa_J61002 (plasmid backbone with RFP) by XS
• P7: Bba_R0040 (ptet) by XP

Digestion during 2h at 37°C.

• Gel migration for P1: pSB2K3, P2: BBa_J61002 (plasmid backbone with RFP), P7: BBa_R0040 (ptet)
• [1kb|P1|P2|P7|100bp|/]

• Result:
  • pSB2K3: wrong bande. Try again...
  • plasmid backbone with RFP: Wrong enzymes?!
  • ptet: good but the 1500bp band must not be there!

• ON culture for S8: pSB2K3, S29: pSB1A3, S30: BBa_K136050 (RBS tetR) in order to done new miniprep

• Resuspention of biobrick BBa_J2479 (1L7)
• Transformation in competent DH5α